This invention relates to methods for inhibiting herpes replication and for treating herpes infection in a mammal by inhibiting the herpes helicase-primase enzyme complex. In a preferred embodiment, this invention relates to thiazolylphenyl derivatives that inhibit the herpes helicase-primase. This invention also relates to pharmaceutical compositions comprising the thiazolylphenyl derivatives, to methods of using and producing the thiazolylphenyl derivatives.
Herpesviruses inflict a wide range of diseases against humans and animals. For instance, herpes simplex viruses, types 1 and 2 (HSV-1 and HSV-2), are responsible for cold sores and genital lesions, respectively; varicella zoster virus (VZV) causes chicken pox and shingles; and the human cytomegalovirus (HCMV) is a leading cause of opportunistic infections in immunosuppressed individuals.
Herpesviruses are complex double-stranded DNA viruses that encode all the enzymes that directly mediate viral chromosomal replication. Seven DNA replication-associated polypeptides are required for human herpesvirus replication. Six of these seven polypeptides show a high degree of homology across all studied human herpesviruses. These six polypeptides, when expressed by the virus, constitute a heterodimeric DNA-dependent DNA polymerase, a monomeric single-stranded DNA binding protein, and a heterotrimeric helicase-primase complex. The seventh DNA replication-associated polypeptide does not display sequence or functional conservation and is involved in the initiation of lytic viral replication.
Without the function of each of the seven herpesvirus-specific DNA replication proteins, herpesvirus chromosomal replication will not initiate or propagate. This has been demonstrated in two ways for DNA replication in HSV-1. First, temperature sensitive HSV-1 strains have been developed and the complementation groups within these strains mapped on a one-to-one correspondence to the seven HSV DNA replication genes. Additionally, transient replication assays that utilized recombinant DNA plasmids containing single DNA replication genes have found that the presence of each of the seven genes was required for the efficient replication of a tester plasmid containing an HSV-1 origin of DNA replication.
More recently, the DNA replication genes in other herpesviruses (i.e., Epstein-Barr virus, cytomegalovirus and varicella zoster virus) have been delineated. These gene sequences were identified as homologous to the HSV-1 DNA replication genes. Furthermore, transient replication assays containing either an Epstein-Barr virus or cytomegalovirus lytic origin of DNA replication confirmed their identity. In varicella zoster virus (the human herpesvirus most closely related to HSV-1) DNA replication genes were found to be highly homologous to HSV-1 ( greater than 50% at the amino acid level) and present at identical relative locations on the two viral chromosomes. Although no follow-up analysis on varicella zoster virus DNA replication genes has been presented to date, it is highly unlikely that differences in the varicella zoster virus and HSV-1 DNA replication programs exist.
From the above, it is clear that human DNA replication proteins are unable to substitute for the HSV-1 encoded enzymes. Otherwise, temperature-sensitive viral polypeptides would have been complemented by human counterparts and the defective viruses would have continued to grow and replicate, even at elevated temperatures. Similarly, in transient replication assays, if human proteins were capable of complementing any of the seven herpesvirus-encoded polypeptides, an absolute dependence on the presence of each of these herpesvirus DNA replication-specific genes would not have been observed. Therefore, inhibiting the activity of those virally-encoded proteins represents an effective way of preventing herpesviral replication.
The helicase-primase enzyme occupies a key and critical place in the herpesvirus DNA replication program. The observation that the genes encoding the herpes helicase-primase are not only essential for replication, but are also highly conserved across the range of known herpesviruses underscores the importance of this enzyme in mediating viral chromosomal replication.
In the helicase-primase complex, two of the three polypeptides (e.g., the expression products of the UL5 and UL52 genes of HSV-1) promote catalysis of duplex DNA unwinding and RNA primer biosynthesis. The third polypeptide, encoded by the UL8 gene, appears to modulate primase activity. The assembled helicase-primase enzyme complex functions both in the initiation and propagation stages of herpesvirus DNA replication. It is responsible for the synthesis of RNA primers necessary for the initiation of all new DNA synthesis by the herpesvirus DNA polymerase. Additionally, for DNA replication to proceed, duplex viral chromosomal DNA must first be unwound to the single-stranded replicative intermediate because the herpesvirus DNA polymerase is inactive on fully duplex DNA. The helicase-primase is also responsible for this important DNA unwinding event.
Conventional anti-herpes therapies have not focused on inhibiting the activity of the herpes helicase-primase(see R. E. Boehme et al., Annual Reports in Medicinal Chemistry, 1995, 30, 139). The most widely used anti-herpes agents to date are purine and pyrimidine nucleoside analogs, such as acyclovir and ganciclovir. These nucleoside analogues inhibit replication of viral DNA by their incorporation into a growing DNA strand. The nucleoside analogue-based inhibitors of HSV-1 growth have found only limited success and are not generally useful in treating recurring infections in the majority of patients. In addition, the infection of humans by other herpesviruses, such as varicella zoster virus or cytomegalovirus, show little or no responsiveness to nucleoside-based therapies.
The lack of broad spectrum anti-herpesvirus activity by the nucleoside-based therapies is not surprising because these compounds act by indirect biological mechanisms. Nucleoside analogues must first be activated to the nucleoside monophosphate by a virally-encoded thymidine kinase enzyme. It should be pointed out that only HSV and varicella zoster virus encode thymidine kinase enzymes. This may, in part, explain the inability to adapt nucleoside-based therapies to the treatment of other human herpesviruses. After initial phosphorylation, the nucleoside analogue monophosphate must be further phosphorylated to the triphosphate by human-encoded enzymes prior to its action. Ultimately, the triphosphorylated nucleoside analogue is incorporated into a nascent DNA chain during viral genomic replication, thereby inhibiting the elongation of that DNA chain by the herpes DNA polymerase.
The final incorporation step of the nucleoside-based therapies has been characterized as xe2x80x9ccompetitivexe2x80x9d because the herpes DNA polymerase does not display a preference for the activated nucleoside drug versus normal deoxynucleoside triphosphates. However, because the action of the DNA polymerase is not considered rate-limiting for herpesvirus DNA replication, the utility of nucleoside-derived compounds in treating herpesvirus infections is necessarily limited. Accordingly, the need for effective, safe therapeutic agents for treating herpesvirus infections continues to exist.
The invention described herein overcomes the above-mentioned limitations and satisfies the above-mentioned needs by providing non-nucleoside-based inhibitors that act directly in interfering with the likely rate-limiting process in herpesvirus DNA replication: the action of the helicase-primase enzyme. Furthermore, since the herpesvirus helicase-primase enzyme is conserved across the human herpesviruses, compounds of this invention are effective against the full spectrum of herpesviruses, including HSV, varicella zoster virus and cytomegalovirus, and also against nucleoside-nonresponsive and nucleoside-resistant herpes infections.
One objective of this invention is to provide methods for inhibiting a herpes helicase-primase, for inhibiting replication of a herpesvirus and for treating herpes infection in a mammal using a non-nucleoside compound characterized by:
(a) an ability to inhibit DNA-dependent NTPase activity of the herpes helicase-primase;
(b) an ability to stabilize the interaction between the herpes helicase-primase and a DNA substrate;
(c) an inability to inhibit DNA-independent NTPase activity of the herpes helicase-primase;
(d) an inability to bind directly to double-stranded DNA; and
(e) an inability to inhibit the herpes origin binding protein helicase encoded by the UL9 gene of HSV.
A further objective of this invention is to provide thiazolylphenyl derivatives useful in the methods of this invention and pharmaceutical compositions comprising those thiazolylphenyl derivatives.
Another objective of this invention is to provide processes for preparing the thiazolylphenyl derivatives of this invention.
Yet another objective of this invention is to provide a method for identifying non-nucleoside herpes helicase-primase inhibitors by screening for (1) inhibition of single-stranded DNA-dependent NTPase activity and (2) lack of inhibition of DNA-independent NTPase activity of a herpes helicase-primase.
A further objective of this invention is to provide non-nucleoside herpes helicase-primase inhibitors identified using the methods of this invention.
Yet a further objective of this invention is to provide non-nucleoside herpes helicase-primase inhibitors characterized by:
(a) an ability to inhibit DNA-dependent NTPase activity of the herpes helicase-primase;
(b) an ability to stabilize the interaction between the herpes helicase-primase and a DNA substrate;
(c) an ability to inhibit replication of a herpesvirus in cell culture by at least about 50% at a concentration of less than about 500 nM;
(d) an inability to inhibit DNA-independent NTPase activity of the herpes helicase-primase;
(e) an inability to bind directly to double-stranded DNA; and
(f) an inability to inhibit the herpes origin binding protein helicase encoded by the UL9 gene of HSV-1.
Still a further objective of this invention is to provide pharmaceutical compositions containing the non-nucleoside inhibitors of this invention and methods for treating herpes infection in a mammal using those pharmaceutical compositions.